Study On The Pharmacokinetics And Metabolomics Of The Main Components Of Atractylodes Japonicus Before And After Processing Based On LC-MS Technology

Purpose: To investigate the difference of pharmacokinetics of the main components between bran fried and raw Atractylodes,and to study what effect can bran fried Atractylodes bring to different endogenous metabolites of spleen deficiency rats,with explaining the mechanism of enhanced efficacy of bran fried Atractylodes from pharmacokinetic and metabolomics perspective.Materials and methods:1.A rapid,specific,sensitive and accurate method was established for the detection of Atractylodes I,Atractylodes II,Atractylodes III and Atractyloside A in rat plasma,The column was Waters ACQUITY UPLC BEH C18(1.7 m,2.1 * 100 mm),the mobile phase was consisted of 0.1% formic acid water(A)-acetonitrile(B),0~1.5 min,5%~10% gradient elution(B),1.5~3 min,10%~25%(B),3~6 min,25%~80%(B),6~8 min,80%,8~8.5min,80%~5%(B),8.5~10 min,5%.The flow rate was 0.3 ml/min,and the column temperature was 40.The sample room temperature was 10?,sample size is 2?l.The detection mode was positive ion multi reaction monitoring mode(MRM),and the electrospray ion source(ESI source)was used.The ionization voltage was 3 KV,the temperature of desolvent gas was400 ?,the rate of degaseous gas flow was 800 L/Hr,the core gas velocity is 150 L/Hr,and the atomized gas pressure was 7 bar.Methodological validation,referring to FDA's guidelines for validation of analytical methods and SFDA's requirements for pharmacokinetic research methodology,examined the precision and accuracy,matrix effect,stability(room temperature stability,repeated freezing and thawing)and other parameters.2.SD rats of SPF grade were used as experimental subjects.The rats were randomly allocated into blank control group,raw Atractylodes group and bran fried Atractylodes group.Samples were collected in 0.083,0.016,0.25,0.5,1,1.5,2,3,4,6,8,10,12 h after oral administrated of Atractylodes extrect.The concentrations of main components in blood were determined by UPLC/MS-MS.After being processed by DAS3.0 program,the pharmacokinetic parametersof these four components were calculated,and the time curves were drawn to clarify whether bran fried can promote the absorption of components and improve their bioavailability.3.The SD rats of SPF grade were selected as the experimental subjects.The rats were randomly divided into normal group,model group,raw Atractylodes group and bran fried Atractylodes group.The model of spleen deficiency was caused by diet,excessive fatigue and bitter cold diarrhea,and enzyme linked immunoassay(ELISA)was used to determine the biochemical indexes of VIP,SS,SPand SDH in each group.The difference of content for VIP,SS,SPand SDH in each group was compared.The metabolites of the spleen deficiency model group and the normal control group were compared with the principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA),and the spleen deficiency differential metabolites were found.4.The effect of raw Atractylodes and bran fried Atractylodes to the spleen deficiency differential metabolites:Combined with OPLS-DA pattern recognition,our study evaluted the overall effect of bran fried for the metabolites of spleen deficiency.Result:1.The experimental results showed that the established UPLC/MS-MS method had high specificity and sensitivity,and could be used for the simultaneous determination of the concentration of atractylodes I,atractylodes II,atractylodes III and atractyloside A in rat plasma after oral administrating of Atractylodes.The peak area and concentration of the four components have a good linear relationship in the concentration range,and precision and stability meet the requirements.2.After oral administrated of raw Atractylodes,the maximum blood concentration of atractylode I,atractylode II,atractylode III and atractyloside A in plasma was 32.088 ± 2.05ng/mL,49.616 ± 7.692 ng/mL,87.041 ± 17.025 ng/mL and 57.80 ±21.65 ng/mL,respectively.After oral administrating of bran fried Atractylodes,the maximum blood concentration of atractylode I,atractylode II,atractylode III and atractyloside A in plasma was 66.94±10.89ng/mL,55.9±13.58 ng/mL,113.10±19.04 ng/mL and 69.38±8.29 ng/mL,respectively.The maximum blood concentration of atractylode I,atractylode II,atractylode III and Atractyloside A increased in various degrees after Atractylodes being bran fried.The results ofintegrated pharmacokinetics had shown that the maximum blood concentration was54.17±7.16 ng/mL and 70.02±16ng/mL respectively after oral administrating of raw and bran fried Atractylodes3.Compared with the normal group,the content of VIP,SS,SP and SDH in the serum of the model group all decreased.Compared with the model group,the contents of VIP,SS,SP and SDH in the serum of raw Atractylodes and bran fried Atractylodes were all promoted,and the effect of bran fried Atractylodes was better than that of the raw Atractylodes.The results of metabolomics study showed that,compared with the normal group,17 kinds of spleen deficiency differential metabolites were selected in the spleen dificiency rats group;nine of these components were found in the positive model,including glycogen,oleic acid,nicotinic acid,phosphatidyl choline,phosphoinositide,dihydrofolate acid,cholesterol,4'-phosphoric acid pantothenic acid sulfhydryl;And eight components were found in the negative mode,namely,palmiyl L-carnitine,ribonamine phosphate,N-palmitic glutamic acid,N-acetyl glucosamine,alanine,L-sorbose,glucosamine-6-phosphoric acid and 5-methyl cytosine.It was found that the mechanism of spleen deficiency was closely related to amino acid metabolism,lipid metabolism and glucose metabolism.4.Both raw Atractylodes and bran fried Atractylodes had a positive regulating effect on the metabolites that had involed;And for dihydrofolate,4 '-phospho-pantothyl ethylamine,phosphatidyl choline,inositol phosphate,oleic acid,palmiyl L-carnitine,ribonylamine phosphate,-6-phosphoric acid,nicotinic acid and alanine.The effect of raw Atractylodes is better than bran fried Atractylodes.Conclusion:1.By comparing the maximum blood drug concentration of Atractylodes I,Atractylodes II,atractylode III and Atractyloside A in raw Atractylodes and bran fried Atractylodes,It founds that the maximum blood concentration of these four components in the plasma of the rat after oral administrating bran fried Atractylodes is higher than that of the raw Atractylodes,which indicates that the bran fried can promote the absorption of the main components in Atractylodes.2.It can be found that both raw Atractylodes and bran fried Atractylodes can regulate the metabolic pathways of unsaturated fatty acids,glucose alanine circulation,palmitic L-carnitine metabolism,glucosamine metabolism,phosphatidylcholine metabolism,and inositol phosphate metabolism,the effect of bran fried Atractylodes is better than that of raw Atractylodes.